Periodic Reporting for period 1 - TheraMatrix (Development of functional, chemically defined coatings for 2D and 3D cell culture applications)
Reporting period: 2021-07-01 to 2023-06-30
Stem cells have the capacity to self-renew and differentiate, which is important for the growth and maintenance of tissues and organs. Thereby, stem cells hold a fundamental promise to advance regenerative medicine, cell therapies as well as cellular agriculture. One of the major challenges of cell-based industries is the production of stem cells at industrial scale. The established cultivation methods in 2-dimensional cell culture articles are not feasible to meet the needs. The cultivation of cells in bioreactors on 3-dimensional (3D) scaffolds/microcarriers is the inevitable future strategy. Currently, there is only a limited number of scaffolds/microcarriers and some of them do not fulfil the performance or safety criteria for stem cells.
In this project, we aimed to develop a universal coating procedure for 3D scaffolds and microcarrier based on our proprietary biomatrix technology. The biomatrix consists of chemically defined, bioresponsive polymers deposited on the surface of scaffolds/microcarriers using a layer-by-layer procedure. We further intended to enhance the understanding of the physico-chemical principles of this molecular deposition procedure.
With the development of such a universal coating procedure and the improved understanding of the underlying mechanisms we aimed to improve the functionality of cell culture scaffolds and microcarriers for bioreactor culture. The optimized growth surface of such scaffolds and microcarriers will ultimately lead to a higher quality and yield of cells and as such address the bottleneck of cell manufacturing.
In this project, we aimed to develop a universal coating procedure for 3D scaffolds and microcarrier based on our proprietary biomatrix technology. The biomatrix consists of chemically defined, bioresponsive polymers deposited on the surface of scaffolds/microcarriers using a layer-by-layer procedure. We further intended to enhance the understanding of the physico-chemical principles of this molecular deposition procedure.
With the development of such a universal coating procedure and the improved understanding of the underlying mechanisms we aimed to improve the functionality of cell culture scaffolds and microcarriers for bioreactor culture. The optimized growth surface of such scaffolds and microcarriers will ultimately lead to a higher quality and yield of cells and as such address the bottleneck of cell manufacturing.
Within the project, various methods were applied to study the interaction of biomatrix polymers with the surfaces of scaffolds/microcarriers. The successful application of the biomatrix was demonstrated by a proof-of-principle study using microcarriers and primary stem cells in a simplified bioreactor culture.
In the first part of the project, a layer-by-layer procedure for the deposition of the biomatrix was analyzed and optimized for stem cell growth on 2D culture surfaces. Analytical methods such as atomic force microscopy (AFM), colorimetric assays and spectrophotometry, fluorescent imaging and zeta potential measurement were applied to study the deposition of the polymers on different surfaces and critical parameters were identified. Our results indicated that charge-based absorption of polymers can be employed to coat various materials applicable for cell culture. Finally, a qualitative model for the biomatrix deposition on cell culture plastic ware was developed. The deposition model was further verified using materials with different surface charge properties.
In the second part of the project, the defined layer-by-layer procedure was transferred to 3D scaffolds and microcarriers. A model microcarrier was used to test the applicability and efficiency of the biomatrix deposition. Subsequently, various scaffolds and microcarriers made of synthetic and natural materials were successfully coated.
Finally, 3D cell culture experiments were carried out for proof-of-concept. Therefore, primary stem cells, in particular mesenchymal stromal cells, were cultured for 10 days in a shaker flask culture system. The results showed that these primary stem cells can be successfully cultured on microcarriers functionalized with the optimized layer-by-layer procedure developed in this project.
The results of this project have the potential for commercial exploitation. The deposition model and obtained data complemented a European patent application. Furthermore, existing products benefited from the development of the deposition model and optimization of coating procedure. In addition, using the optimized layer-by-layer procedure will drastically shorten the development periods of new products. In the future, the host organization plans to bring a novel microcarrier to the market that will enable improved bioreactor culture and higher cell numbers to meet the demands of cell-based industries.
In the first part of the project, a layer-by-layer procedure for the deposition of the biomatrix was analyzed and optimized for stem cell growth on 2D culture surfaces. Analytical methods such as atomic force microscopy (AFM), colorimetric assays and spectrophotometry, fluorescent imaging and zeta potential measurement were applied to study the deposition of the polymers on different surfaces and critical parameters were identified. Our results indicated that charge-based absorption of polymers can be employed to coat various materials applicable for cell culture. Finally, a qualitative model for the biomatrix deposition on cell culture plastic ware was developed. The deposition model was further verified using materials with different surface charge properties.
In the second part of the project, the defined layer-by-layer procedure was transferred to 3D scaffolds and microcarriers. A model microcarrier was used to test the applicability and efficiency of the biomatrix deposition. Subsequently, various scaffolds and microcarriers made of synthetic and natural materials were successfully coated.
Finally, 3D cell culture experiments were carried out for proof-of-concept. Therefore, primary stem cells, in particular mesenchymal stromal cells, were cultured for 10 days in a shaker flask culture system. The results showed that these primary stem cells can be successfully cultured on microcarriers functionalized with the optimized layer-by-layer procedure developed in this project.
The results of this project have the potential for commercial exploitation. The deposition model and obtained data complemented a European patent application. Furthermore, existing products benefited from the development of the deposition model and optimization of coating procedure. In addition, using the optimized layer-by-layer procedure will drastically shorten the development periods of new products. In the future, the host organization plans to bring a novel microcarrier to the market that will enable improved bioreactor culture and higher cell numbers to meet the demands of cell-based industries.
The project advanced the understanding of the charged-based interaction of polymers with surfaces. Therewith, products with improved performance and quality can be developed for the culture of stem cells. These products can be produced more efficiently through reduced material and energy input and at the same time result in higher cell yields. These aspects will pay on the account towards establishing stem cell culture for therapeutic or nutritional purposes with relevant socio-economic impact.